MODX LAB MIDTERMS

• Gel electrophoresis is the standard lab procedure for seperating DNA by size (e.g., length in base pairs) for visualization and purification.
• Principle: Electrophoresis uses an electrical field to move the negatively charged DNA through an agarose gel matrix toward a positive electrode.
• Black wire: Negatively charged (CATHODE)
• Red wire: Positively charged (ANODE)
• Shorter DNA fragments migrate through the gel more quickly than longer ones.
• You can determine the approximate length of DNA fragment by running it on agarose gel alongside a DNA ladder.
• DNA ladder is a solution that contains known length of DNA fragments
• Well combs is used to create wells in the gel matrix.
• The distance between tooth of the well comb is 0.05 mm to 4 mm
• Voltage source requires 80-150 volts but is usually need a 100 volts
• Gel chamber is made up of acrylic (non-conductive)
• Bands are visualized by gel documentation immediately to prevent destruction of DNA due tov prolong exposure to UV light
• Agarose gel powder melts at 80C, cooling effect at 55-65C, and solidify at 32-40C
• DNA stain

  SyBr stain: binds to the minor groove of the DNA

  Ethidium bromide: mutagenic; intercalates with the DNA
• DNA ladder is used to identify DNA base pairs length; it contains known base pairs length
• Tracking dye is used to monitor migration through the gel and is usually seen in the loading buffer mixed with the sample
• Sedimentation agent allows the DNA solution to settle at the bottom of the well
• Examples of sedimentation agents are:

  1. Glycerol/Glycerine
  2. Sucrose/Sucralose (artificially made)
  3. Ficoll
• Tris-Acetate-EDTA running buffer is the fastest migration since it conducts more electricity
• Tris-Acetate-EDTA running buffer is not suitable for longer runs since there's a chance for it to migrate beyond the gel
• Tris-Borate-EDTA running buffer has higher buffering capacity that maintains pH despite electrical flow
• Tris-Borate-EDTA running buffer is commonly used for better seperation of small DNA fragments
• Tris-Borate-EDTA running buffer has higher ionic strength makes it suitable for long runs
• TBE buffer is prone to precipitation that creates salt waves (local distortion of nucleic acids)
• Procedure in pouring a standard 1% agarose gel

  1. Measure 1 g of agarose

  2. Mix agarose powder with 100 ml 1xTAE in a microwavable flask

  3. Microwave for 1-3 mins until the agarose is completely dissolved (do not overboil!)

  4. Let agarose solution cool down to about 50C (takes about 5 minutes)

  5. Add the DNA stain to the melted agarose

  6. Pour the agarose into the gel tray with the well comb in place

  7. Place newly poured gel at 4C for 10-15 minutes or let sit at room temperature for 20-30 minutes, until it has completely solidified

  8. Place in the gel box

  9. Add buffer

  10. Put bleach on the 1xTAE then discard in sink
• The buffer will evaporate = alter the final percentage of agarose in the gel
• Microwave procedure


  Microwave for every 30-45 seconds, stop and swirl, and then continue towards a boil
• Run the gel at 80-150 V (100V) until the dye is approximately 75-80% of the way down the gel.
• A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage
• The fragments of DNA are usually referred to as bands due to their appearance on the gel
• If you will be purifying the DNA for later use, use long wavelength UV and expose for as little time as possible to minimize damage to the DNA
• Using a semi-log graph, plot the bands of DNA ladder and its distance to create an average curve
• How to get a better resolution (crispness) of the bands?

  1. Run the gel at a lower voltage for a longer period of time
  2. Using a wider/thinner gel comb
  3. Loading less DNA into the well (100-150 ul)
  4. Choose the optimal agarose concentration
• Gel percentage: 0.5

  Range of efficient separation: 2,000-50,000 (longest base pair)
• 5:1

  5 volume of sample

  1 volume of buffer
• For small gels: 8x10 cm gels (30-50 ml)

  For larger gels: Southern and northern blotting (250 ml)
• "Uneven heating of the gel across different lanes caused by high voltage"

  Remedy: reduce the voltage
• "Uneven distribution of the electric field across the gel width"

  Remedy: check the tank setup for loose contacts/electrode
• 3-5 mm of buffer covering the gel's surface to prevent distortion; Excess buffer = band distortion
• Commonly encountered problems:

  1. Faint bands = fuzzy decreased sample
  2. Smeared bands = blurry /diffused
  3. Poorly separated bands = stacked appearance
  4. Smiling bands
  5. Incorrect quantification = incorrect loading dye or buffer